A new validated HPLC-UV method for therapeutic monitoring of daptomycin in comparison

with reference Mass Spectrometry

Giacomo Luci1,#,*, Federico Cucchiara1,#, Laura Ciofi2, Marianna Lastella2, Romano Danesi1,2,

Antonello Di Paolo1,2

Affiliations

1, Department of Clinical and Experimental Medicine, University of Pisa, Via Roma 55, 56126,

Pisa, Italy.

2, Unit of Clinical Pharmacology and Pharmacogenetics, University Hospital, Via Roma 55, 56126,

Pisa, Italy.

# = The two authors equally contributed to the paper

* = Corresponding Author:

Giacomo Luci

Department of Clinical and Experimental Medicine

University of Pisa

Via Roma 55, 56126, Pisa, Italy

Tel: +39 0502218755

E-mail: giacomo.luci@for.unipi.it

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Abstract

Daptomycin, a cyclic lipopeptide antibiotic with a broad spectrum of activity against Gram-positive

bacteria, is also active against multi-resistant bacterial strains, as well as methicillin-resistant S.

aureus, vancomycin-resistant enterococci or penicillin-resistant S. pneumoniae. For these reasons it

is a viable alternative for the treatment of persisting infections. However, the therapeutic drug

monitoring of daptomycin is recommended because the known variability in drug disposition and

the severe clinical conditions of patients. Therefore, we developed a simple and fast UV-HPLC

method according to FDA guidelines to monitor plasma concentrations of the drug. Briefly, after a

liquid-liquid extraction, plasma calibration samples, quality controls and patients’ samples were

injected in a HPLC instrument and peaks of daptomycin and gentamicin (internal standard) were

resolved by a C18 250 x 4.6 mm, 5 µm stationary phase and peaks were monitored at UV=262 nm.

Mobile phase (isocratic flow of 1 mL/min) consisted of acetonitrile-buffer (KH2PO4 20 mM

pH=3.2) 46:54, vol/vol. Under these conditions, IS and daptomycin peaked at 4.1 and 5.8 min after

injection. Values of limits of detection and quantitation accounted for 1.65 and 5.00 (g/ml),

respectively. Values of method linearity (r2) in range 5-100 mg/L were 0.9975 and 0.9956 plasma

samples and solvent standard, respectively. Inter- and intra-day variability coefficients were lower

than 15%. The comparison with a reference, commercially-available LC-MS/MS method on 122

patient plasma samples returned excellent correlation (r2=0.9474). In conclusion, the present

method demonstrated to be reliable and suitable for daptomycin TDM in clinical routine.

Keywords

Daptomycin; HPLC-UV; Plasma; Chromatography; Automatic integration; TDM

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Highlights

Daptomycin displays a non-negligible pharmacokinetic variability among patients

Therapeutic drug monitoring may reduce interpatient variability

A robust HPLC-UV method was developed and validated in comparison with a reference

LC-MS/MS method

The availability of such a method may improve daptomycin efficacy and tolerability

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1. Introduction

Daptomycin is one of cyclic lipopeptide antibiotics drugs, with broad spectrum against gram-

positive bacteria [1] that are responsible for severe and difficult-to-treat infections, characterized by

high mortality rate in hospital settings. [2]. For these reasons, the antibiotic has been increasingly

used to treat skin infections, [3], bacterial endocarditis [4], as well as infections caused by

methicillin-resistant S. aureus (MRSA) [5] and vancomycin-resistant enterococcus (VRE) [6].

Although the standard doses ranged from 4 to 6 mg/kg, in several cases physicians are prescribing

higher dosages up to 10 or 12 mg/kg [7]. Daptomycin is characterised by linear pharmacokinetics in

healthy volunteers, and with a plasma half-life of about 9 h, while drug excretion occurs mainly via

the kidneys as parent compound [8]. Only a small fraction of the drug (approximately 3%) is

excreted into the bile [5,9]. However, some factors may influence daptomycin pharmacokinetics

[10]. For example, the dose adjustment is required in patients with chronic kidney disease stages 4

and 5 (glomerular filtration rate, GFR <30 ml/min) [10]. Moreover, the renal replacement therapies

eliminates only a minimal part (about 15%) of drug, depending on the dialysis method applied and

the high plasma protein binding of about 92% [11].

In turn, these alterations can cause changes in plasma concentrations and, consequently, the

occurrence of adverse reactions, such as muscle toxicity with possible rhabdomyolysis, acute renal

failure [12], and hepatotoxicity [13] that could be exaggerated by other drugs [14]. Due to the

severity of these adverse reactions, the therapeutic monitoring (TDM) of drug plasma

concentrations is important to personalize the dosage. Indeed, it has been demonstrated that

daptomycin pharmacokinetics is variably changing according to the severity of the infection and

diseases, with significant changes over time [15] that justify the adoption of TDM protocols

[16,17].

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In recent years, the improving knowledge and technical possibilities have significantly increased the

development of chromatographic methods to identify and quantitate toxic substances, as well as

residues of xenobiotics in food and drugs [18–20]. In particular, new antibiotics as well as

daptomycin have become increasingly prescribed given their efficacy in the presence of resistant

bacteria and difficult-to-treat infections, despite their non-negligible risk of potentially severe toxic

effects. Therefore, the availability of a chromatographic method for therapeutic drug monitoring

(TDM) has been perceived as a need for physicians and laboratory staff. The literature reports

different methods based on mass spectrometry [21], UPLC-PDA [22] and UPLC-UV [23], which

are characterized by high costs and need high degrees of sample purification in order to solve

instrumental problems. In the present study, we describe the development and validation of a new

chromatographic method for the measurement of daptomycin plasma concentrations according to

for Industry FDA guidelines [24]. Moreover, an automatic detection and quantification of peaks

without user input was developed (patent pending). Finally, the validated method was compared

with a liquid-chromatography-mass spectrometry (LC-MS/MS) method that is commercially

available.

2. Materials

2.1 Chemicals

Acetonitrile (CH3CN), phosphoric acid 85% (H3PO4 85%), trichloroacetic acid (TCA), water and

methanol (CH3OH), all reagents of HPLC grade, were purchased from Merck (Merck, Darmstadt,

Germany). Potassium phosphate (KH2PO4), daptomycin and gentamicin sulfate (internal standard)

were purchased from Merck. The Mass spectrometry (LC-MS/MS) kit for the measurement of

antibiotic concentrations in plasma was purchased from Eureka-Lab Division (Eureka One, Ancona,

Italy) and considered as the reference method.

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2.2 Instrumentation

The HPLC-UV method was developed using a Waters 2695 Separations Module equipped with a

Waters 2487 Dual Absorbance Detector (Waters Corporation, Milford, CT) and controlled by the

Empower software version Pro (Waters Corporation). HPLC separation was accomplished on a

HAISIL HL C18 250 mm x 4.6 mm x 5 m (Higgins Analytical Inc., USA). Moreover, plasma

samples were assayed for daptomycin concentrations by the reference LC-MS/MS method using an

Acquity UPLC Binary Solvent Manager pump with Sample Manager autosampler equipped with a

Triple Quadrupole Detector (TQD, Waters Corporation). LC-MS/MS system was controlled by

MassLynx software (version V4.1, Waters Corporation, USA).

Finally, MATLAB R2016b with the bioinformatic toolbox (MathWorks Inc., USA) was used to

process data.

2.3 Methods

2.3.1 Calibration and quality control samples

A daptomycin stock solution was prepared by dissolving 10 mg of daptomycin in 10 ml of water

(final concentration, 1000 g/ml). Calibration and quality control samples were obtained by serial

dilution of drugs (from stock solution aliquots) in human plasma, obtained from healthy volunteers.

From this stock solution, 100 l was diluted with 900 l of blank human plasma (obtained from

healthy volunteers), obtaining a working solution of 100 g/ml. Serial dilutions for calibration

standards were made in blank plasma up to final concentrations of 5, 10, 25, 50, 100 g/ml. For

method validation and intra/inter-day variability the following concentrations were used 5, 50, 100

g/ml. Internal standard validation was performed on gentamycin spiked plasma samples.

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Daptomycin and gentamicin stock solutions (in water) were stored at -20 °C for maximum 7 days,

while spiked plasma was conserved at -20 °C for 3 consecutive days for intra- and inter-day

validation [23].

2.3.2 Sample preparation

To 200 l of quality control, calibration or patient samples, 20 l of gentamicin (IS) 100 g/ml

were added, then samples were vortexed for 30 sec. Samples were treated to remove proteins by

adding different chemicals (i.e., , CH3OH, etc.) and the final choice was based on percentage of

absolute recovery. Samples were vortexed for 30 sec and centrifuged at 7700 g for 15 min; the clear

supernatant was transferred into HPLC autosampler vials for HPLC-UV analysis. Sample

preparations for LC-MS/MS analysis were made according to Eureka® KIT protocol.

2.3.3 HPLC-UV and LC-MS/MS conditions and detection

The HPLC mobile phase consisted of organic solvent (i.e., CH3CN and/or MeOH) plus buffer

KH2PO4 20 mM pH=3.2. The choice of final pH was dependent on the chemical structure of

daptomycin and gentamycin in order to optimize the interaction of analytes with stationary phase.

Moreover, in order to expedite the chromatographic runs, an isocratic elution of the mobile phase

was chosen with chromatographic column maintained at controlled temperature (35 °C). Finally,

because of the chromatographic column size (250 mm x 4.6, 5 µm), the volume of injection was

fixed at 50 l.

Samples analysis by LC-MS/MS was made according to Eureka® Kit procedure and parameters

setting [25].

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2.3.4 Validation studies

The method validation was made according to FDA guidelines through the evaluating of precision

and accuracy. In particular, a limit for intra-day precision was defined as 2(1-0.5logConc)2/3, while a

limit for inter-day precision was calculated as 2(1-0.5logConc). Accuracy was the percentage of results

not deviating more than 15% of nominal concentrations and 20% at LOQ. Quality control

samples were analysed in triplicate at each of the three concentration levels. Samples were

quantified in a single batch, considering mean concentration, standard deviation and percentage

coefficient of variation. Inter-day precision and accuracy were calculated with the same parameters

and quality controls, in triplicate at three different concentrations for three consecutive days. To

evaluate specificity, blank samples of human plasma were analyzed to check for the presence of

interfering peaks at the elution time points of daptomycin and gentamicin. Potential interferences

caused by endogenous and chemically-related compounds, were studied to evaluate the selectivity

of the method and for internal standard. The signal-to-noise ratio of a possible interfering peak in a

blank plasma sample should be below the signal-to-noise ratio of daptomycin and gentamicin in the

same elution sector at the limit of detection (LOD) level. The LOD was evaluated as a signal-to-

noise ratio ≥ 3 while limit of quantification (LOQ) was calculated as 3.04LOD.

The last step of method validation did concern every possible interference by drugs co-administered

with daptomycin. To do this, drugs other than daptomycin were selected on the basis of

concomitant therapies prescribed in patients.

2.3.5 Application of the method and comparison

From November 2018 to February 2019, blood samples from hospital units (i.e., infectious diseases,

cardiovascular diseases, orthopaedics, general medicine and neurosurgery) were dispatched to TDM

laboratory of the Clinical Pharmacology Unit for daptomycin monitoring. Samples were rapidly

processed to measure plasma drug concentration by the commercially-available LC-MS/MS method

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(Eureka One, Ancona, Italy) on the Waters TQD instrument (Waters, Milford, CT). After the

completion of laboratory activities, aliquots of plasma samples (1 mL) were stored at -80 °C for

further processing by using the developed method. The collection and analysis of samples were part

of the DAPTOLIN protocol [protocol number 55945, Pisa University Hospital Ethics Committee],

but none of patients’ information nor clinical data were registered or used for the present work. Raw

results were then analysed by a custom-made MATLAB method® – patent pending (MatLab®, The

Math Works Inc, USA) in order to obtain retention times and area values of analytes peaks. The

linear correlation of peak area values obtained by the developed HPLC-UV method and the LC-

MS/MS kit was investigated.

2.3.6 Statistical calculations

Statistical calculations were performed with Graph Pad Prism version 5.0a (Graph Pad Software®,

USA). Correlation analysis were done between daptomycin concentrations of HPLC-UV data and

LC-MS/MS reference method, that was performed to evaluate the significance of equivalence.

Level of significance was set at p < 0.05.

3. Results

3.1 Sample extraction and HPLC-UV analysis

The sample preparation procedure was chosen after various tests, with same volumetric ratios,

vortexing and centrifugation times. The following solvents were tested for preanalytical preparation

of plasma samples: CH3CN - H3PO4 85% (95:5 v/v), CH3CN, water - TCA (90:10 v/v) and CH3OH.

For every experimental condition, 10 human plasma samples from healthy volunteers were spiked

with daptomycin and gentamicin then they were analysed. Protein precipitation was obtained by

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adding 200 L of each solvent/mixture to 200 L of spiked human plasma. Table 1 reports the

average percentage recovery of plasma samples extracted in different conditions. The highest

recovery of daptomycin and gentamicin was obtained with CH3CN - H3PO4 85% (95:5 v/v).

Table 2 and Figure 1 report retention times of analytes with different mobile phases made by

CH3CN and phosphate buffer KH2PO4 20 mM, pumped with a flow of 1 mL/min in isocratic mode.

The best separation of peaks with a short chromatographic run (i.e., ≤10 min) was obtained with a

mobile phase consisting of CH3CN-phosphate buffer KH2PO4 20 mM pH=3.2 46:54, vol/vol.

Furthermore, temperature of column oven and UV wavelength were set at 35 °C and 262 nm,

respectively. With these chromatographic conditions, the run time was 10 min while daptomycin

and gentamycin have a retention time of 4.1 and 5.8 minutes, respectively. Representative sample

chromatograms with analytes extracted from a human plasma sample are shown in Figure 2.

Noteworthy, average recovery from plasma samples was > 95% for both daptomycin and

gentamicin.

3.2 Validation studies

Validation parameters are reported in Tables 3 and 4, and they completely fulfilled the requirements

of the FDA 2018 analytical parameters guideline [24]. The method was proven to be linear in the

full range of 5-100 mg/L in both plasma samples (r2 > 0.9975) and solvent standards (r2 > 0.9956).

The intra- and inter-day variability values were <12% and < 15%, respectively, at three different

concentrations. The LOQ and LOD values of the method were 5.00 and 1.65 mg/L, respectively.

The results of the selectivity study showed no interferences between endogenous compounds and

the analysed compounds (daptomycin and gentamicin). The average internal standard response in

samples was +3.45% compared to average internal standard response of calibrators. The analysis of

more than 30 different drugs (Table 5) suggested only one probably interference between

daptomycin peak (RT = 4.1 min.) and piperacillin.

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Accuracy and precision values were within acceptable range of variability with respect nominal

concentrations at 5-50-100 mg/L (15%) and LOQ (20%) as per FDA guidelines (Tables 3 and

4).

Finally, the LOD value and calibration range were acceptable on the basis of TDM routine and

therapeutic range. Indeed, the efficacy of daptomycin is associated with maximal plasma

concentrations (Cmax) higher than 60 mg/L, while the risk of drug-associated toxicities increases

when minimum plasma concentrations (Cmin) are higher than 24 mg/L. In particular, only 5 pre-dose

samples had daptomycin concentrations lower than 5.00 mg/L, whereas drug concentrations higher

than 100 mg/L were measured in 2 samples (i.e., 103 and 109 mg/L). Interestingly, the

corresponding Cmin values were 30.8 and 34.5 mg/L.

3.3 Comparison between HPLC-UV and LC-MS/MS analyses on plasma samples and

possible interfering drugs

In order to establish the reliability of the developed HPLC-UV method, the latter was compared

with a LC-MS/MS reference method by measuring daptomycin concentrations in 122 human

plasma samples using both methods. When HPLC-UV results were plotted against those obtained

with mass spectrometry, the correlation analysis returned values of highly statistical significance,

with a r2 value of 0.9474, a slope of 1.052 and a y-intercept of 0.8543 ± 0.9368 mg/L (Figure 3).

Moreover, the Mann-Whitney test and unpaired t test with Welch’s correction returned not

significant differences between results obtained by the two techniques (p values, 1.000 and 0.9927,

respectively).

4. Discussion

The presented manuscript describes the development and the validation of an HPLC-UV method for

the determination of daptomycin in human plasma. The method was then applied to measure

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daptomycin concentration in 122 plasma samples from patients treated with the drug. The findings

showed that the method is precise and reproducible, being able to quantitate plasma concentrations

of daptomycin within the minimum-maximum range of values that are expected after the

administration of the drug at the prescribed doses (4-6 mg/kg/day i.v.). Furthermore, to our

knowledge this is the first HPLC-UV method for daptomycin that has been compared with a LC-

MS/MS reference method. For these reasons, the method is currently used to monitor all plasma

samples dispatched to our Clinical Pharmacology Unit.

The need for therapeutic monitoring of daptomycin is based on the severity of infections that

require the drug and on the interindividual variability observed in pharmacokinetics across several

physio-pathological conditions [26] , as well as the influence of renal function [27]. Therefore, the

optimization of drug dosage within the first days is a fundamental prerequisite to improve cure rate

[27], to diminish the risk of severe toxicities [12,13] and to shorten the stay in intensive care or

infectious disease units. Our method allows the measurement of daptomycin concentration in

plasma samples adopting a liquid-liquid extraction procedure, which is advantageous for the highest

recovery achieved and reduced time needed to sample preparation. The latter characteristic, together

with a 10-min chromatographic run, does ensure the analysis of at least 5 samples per hour. The

sample rate is lower than that of LC-MS/MS methods, however the HPLC-UV platform is simpler

from a technical point of view and less expensive than the mass spectrometry. In terms of

sensitivity, our method has a LOQ of 5.00 mg/L, which is well above the LOQ of a corresponding

LC-MS/MS method. However, TDM of daptomycin is based on two pharmacokinetic endpoints,

significantly associated with efficacy and tolerability. In particular, Cmax values should be higher

than 60 mg/L to achieve a Cmax/MIC (Minimal Inhibitory Concentration) ratio of at least 100 based

on the clinical breakpoint values for daptomycin in sensitive species [28] to reduce the selection of

resistant clones [29]. In our series of patients’ samples, we observed only 2 concentrations higher

than 100 mg/L. Moreover, Cmin values should be lower than 24.3 mg/L to reduce the risk of adverse

drug reactions, especially skeletal muscle toxicity [26,30]. Interestingly, the two samples with the

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highest drug concentrations were correlated with Cmin values higher than 24.3 mg/L, hence

suggesting that TDM of both peak and through samples is appropriate to identify patients at risk of

severe toxicities. Based on these assumptions and observation, the measurement of daptomycin

concentrations lower than the LOQ in 6 samples was not considered an issue for the validation of

the present HPLC-UV method. Therefore, the linearity of the present method in the range 5.00-100

mg/L well encompasses the full window of plasma concentrations measured in patients who

received daptomycin at doses of 4-6 mg/kg/day i.v. or higher [27], while the accuracy and precision

further strengthen the reliability of the present method.

In conclusion, we developed a reliable and rapid HPLC-UV method for the measurement of

daptomycin concentrations in plasma samples using an internal standard for better accuracy and

precision over the range of drug concentrations expected after the administration of daptomycin at

standard doses. Moreover, the simple preanalytical preparation of samples and the reduced costs of

HPLC platform certainly ensure a wide diffusion of the present method.

Conflict of Interest

The authors haven’t conflict of interest.

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References

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Legends of Figures

Figure 1. Analytes retention time. Changes in retention time of analytes (daptomycin and

gentamicin) at different percentages of acetonitrile and phosphate buffer (KH2PO4 20 mM). The pH

of the mobile phase (MP) was 3.2 and it was pumped within the HPLC system at flow of 1 mL/min.

The temperature of column oven was set at 35 °C.

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Figure 2. Chromatograms. Sample chromatograms obtained from extracted plasma samples. A,

blank plasma sample, without interfering peaks detected at retention times of daptomycin (RT=4.1

min) and gentamicin (RT=5.8 min). B and C, chromatograms from plasma samples spiked with

gentamicin and daptomycin together with gentamicin, respectively.

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Figure 3. Methods linear correlation. Linear correlation of daptomycin concentration values

obtained with HPLC-UV and LC-MS/MS methods in 122 plasma samples. The linear correlation

analysis returned a r2 value of 0.9474 (p < 0.0001)

Table 1. Tests with different solvents for plasma proteins precipitation. Summary of percentage

recovery of 10 human plasma samples spiked with daptomycin and gentamycin and extracted with

different solvents.

SolventsPercentage recovery(mean SD, n = 10)

CH3CN - H3PO4 85% (95:5 v/v) 98.2 4.57

CH3CN 85.1 5.47

CH3OH 70.6 3.67

TCA 10 % 45.9 4.65

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Abbreviations: TCA, trichloroacetic acid

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Table 2. Tests with different mobile phases to obtain non-overlapping peaks of daptomycin and

gentamicin. The pH of mobile phase was 3.2 and the chromatographic run was 10 minutes

Mobile phase (% v/v)KH2PO4, 20mM – CH3CN

Optimal separation of analytes

60 - 40 No

55 - 45 No

54 - 46 Yes

50 - 50 No

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Table 3. Inter-day validation parameters. The inter-day parameters were calculated on 3

consecutive days and 6 set of samples per day. Measured concentrations are meanSD values

Concentration (mg/L)Accuracy

(%)Precision

(%)CV(%)DAY Theoretical Measured

1

5 4.77 ± 0.70 4.64 0.88 14.61

50 49.32 ± 2.26 2.20 0.20 0.00

100 98.29 ± 2.78 1.71 0.05 2.83

2

5 4.77 ± 0.69 4.58 0.88 14.39

50 48.80 ± 2.26 2.40 0.21 4.64

100 97.76 ± 3.99 2.24 0.01 4.08

3

5 4.62 ± 0.46 1.41 0.87 0.00

50 50.41 ± 3.34 -0.81 0.20 6.62

100 99.75 ± 0.76 0.25 0.001 0.77

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Table 4. Intra-day validation parameters. The intra-day parameters were calculated on 6 set of

samples per day. Measured concentrations are meanSD values. The table also reports the limits

of detection (LOD), quantitation (LOD) and r 2 of linear regression analysis for daptomycin spiked in

blank human plasma samples.

Concentration (mg/L)Accuracy

(%)Precision

(%)CV(%)

LOD (mg/L)

LOQ (mg/L)

r2

Theoretical Measured

5 4.77 ± 0.83 3.19 1.32 11.65

1.65 5.00

0.9975

±

0.0009

50 49.74 ± 2.39 0.53 0.30 4.80

100 98.60 ± 2.86 1.40 0.01 2.90

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Table 5. Drugs that do not interfere with the identification and quantitation of daptomycin and

gentamicin peaks in chromatograms. Piperacillin/tazobactam may be able to alter

chromatographic peaks.

Drugs not interfering with the identification and quantitation of daptomycin and gentamycin peaksA - Acamprosate, Acetil salicylic acid, Aldactone, Allopurinol, Alprazolam, Amiodarone

B - Bisoprolol, Bromazepam

C - Canrenoate potassium, Canrenone, Carvedilol, Clindamycin, Clopidogrel, Carbidopa,

Codeine, Colchicine

D-F - Delorazepam, Diazepam, Enoxaparin sodium, Entecapone, Fluconazole, Furosemide

I - Isosorbide mononitrate

L-O - Lansoprazole, Levodopa, Levothyroxine, Linezolid, Lorazepam, Omeprazole, Oxacillin,

Oxycodone

P-R - Pantoprazole, Prednisone, Ramipril, Rifampicin

S-T-W - Sertraline, Sulfamethoxazole, Tamsulosin, Tazobactam, Teicoplanin, Tigecycline,

Triazolam, Trimetoprim, Warfarin

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